Hypoxia and nutrient deprivation conditions are dynamic features of the tumor microenvironment that contribute to cancer progression and resistance to treatment. We have previously shown that hypoxic stress activates the endoplasmic reticulum (ER) kinase PERK thereby inducing phosphorylation of the translation initiation factor eIF2? on ser51. Later, we also demonstrated that the nutrient-sensing kinase GCN2, similarly activates eIF2 phosphorylation in solid tumors in response to both amino acid and glucose deprivation. Phosphorylation of eIF2? not only reduces energy expensive processes such as global translation, but also creates an environment that promotes the more efficient translation of stress-responsive genes, such as ATF4, a transcription factor that upregulates genes involved in adaptation to ER stress. The phosphorylation of eIF2? and the upregulation of ATF4 represent a common mechanism activated by different cellular stresses, thereby being termed the Integrated Stress Response (ISR). Disruption of the ISR in tumor cells dramatically affects their proliferation and survival under stress and their ability to grow tumors in vivo. Together, our data support a model in which transformed cells activate the ISR in vivo as an adaptive response to oxygen and nutrient deprivation stress and that disruption of this pathway at several steps compromises cellular survival under stress and tumor growth. The overall hypothesis of this proposal is that the ISR transducers PERK and GCN2 which are activated under conditions of tumor microenvironmental stress, activate pathways that lead to increased cell survival and angiogenesis and contribute to metastasis. To test this hypothesis, we propose the following three specific aims: In Aim 1, we will determine the role of the cyclin-dependent kinase inhibitor p21 in mediating cell-cycle arrest and survival in response to hypoxia and nutrient deprivation in ISR-proficient and deficient cells. In Aim 2, we will investigate the role of GCN2 and PERK in angiogenesis using in vitro angiogenesis models. We will also identify mediators of angiogenesis downstream of GCN2 and PERK using antibody arrays and sucrose sedimentation analysis of actively translated mRNAs. In Aim 3, we will use transgenic mouse models of fibrosarcoma which will be crossed to GCN2+/+ and GCN2-/- mice. Angiogenesis and metastasis will be investigated in these models. Completion of these aims will establish whether the ISR is a critical targets of tumorigenesis and metastasis and define the mechanism of such an activity. Inhibitors of PERK and GCN2 are being actively pursued by the PI's lab and by pharmaceutical companies. Therefore, such data could facilitate rapid movement of lead compounds into preclinical animal testing phase. PUBLIC HEALTH RELEVANCE: The tumor microenvironment plays important roles in making tumors more aggressive and more resistant to therapy. This proposal continues work performed over the last 10 years to understand the molecular mechanisms by which these stresses (such as low oxygen and low glucose) increase the ability of tumors to make new blood vessels, grow and spread to other sites. Successful completion of the aims in this proposal may lead to new targets for therapeutic intervention against aggressive cancers such as fibrosarcomas.